Department of Urology, New York University School of Medicine, New York, New York.
Veterans Affairs New York Harbor Healthcare System, Manhattan Campus, New York, New York.
Department of Pathology, New York University School of Medicine, New York, New York.

Introduction

Muscle-invasive urothelial carcinoma of the bladder (MIUCB) is amongst the most aggressive and deadliest cancers (1). Because of its high risk of progression to metastatic stages, MIUCB often calls for multiagent neoadjuvant chemotherapy followed by radical cystectomy or adjuvant chemotherapy after the surgery or radiotherapy concomitant with systemic chemotherapy (2, 3). Despite such debilitating therapies, over 50% of MIUCB advance to local and distant metastasis, at which point the 5-year survival rates are only about 30% and 5%, respectively (1).

A significant recent development is the recognition that MIUCB is not a single disease entity but comprises distinct subtypes distinguishable by combinatorial molecular signatures and divergent clinical outcomes (4–11). While the exact number, interrelationship, and spectra of the molecular signatures between different subtypes from different studies remain to be delineated, a consensus is emerging pointing to at least two major subtypes: luminal and basal. The luminal subtype bears features of the luminal umbrella cells of normal urothelium, for example, high levels of uroplakins, cytokeratin 20, and E-cadherin (4–6, 9–11). Mutations of fibroblast growth factor 3 (FGFR3) and tuberous sclerosis 1 (TSC1) are prevalent along with alterations involving many other genes. The basal subtype, on the other hand, expresses abundant proteins associated with the basal cells of normal urothelium, such as cytokeratins 14, 5, and 6B as well as markers signifying increased stemness and epithelial-to-mesenchymal transition (EMT; e.g., high CD44, TWIST1/2, SNAI2, ZEB2, VIM, and N-cadherin and low E-cadherin and claudin; refs. 4–6, 9, 10). Focal squamous differentiation is common in this subtype and, as suspected, the basal subtype is much more aggressive and correlates with more advanced stage and poorer prognosis than is the luminal subtype (4, 8, 10). Notably, the frequency of p53 mutations that characterize MIUCB in general does not differ significantly between the two major subtypes, although one study found RB1 pathway alterations to be more prevalent in the basal subtype than in the luminal subtype (5).

Notwithstanding the recent progress in subtyping MIUCB, several critical issues remain. First and foremost, are different subtypes of MIUCB caused by distinct genetic drivers? Thus far, most subclassification studies are based on expression signatures including those of uroplakins, cytokeratins, and cadherins (9–11), which are not genetic tumor drivers but phenotypic consequences of urothelial differentiation vis-à-vis dedifferentiation. Those making use of gene mutations for subclassification often involve multiple alterations (8) whose relationship with a particular subtype remains correlative. A definitive cause–consequence effect between a minimum essential set of genetic drivers and a given subtype requires experimental verification using biologically relevant systems. Such biologic studies are important because defining the genetic driver(s) could not only simplify the subtyping of MIUCB and reduce the number of prognosticators, but also narrow down druggable targets for precise therapeutic intervention (12). Second, do different subtypes of MIUCB progress via divergent phenotypic pathways? Clinicopathological studies have long held that MIUCB can (i) arise de novo (i.e., without a defined precursor), (ii) progress from flat, carcinoma in situ (CIS) precursor lesions, or (iii) progress from high-grade, noninvasive papillary urothelial carcinomas (13–16). It is crucially important to determine whether some of the MIUCB subtypes are actually a result of tumor progression from a particular premalignant lesion, so that specific strategies can be devised to predict and prevent progression. Third, do different MIUCB subtypes originate from different normal urothelial cell types? Normal urothelium can be divided into at least three different compartments: basal, intermediate, and luminal (17). Although all urothelial carcinomas were previously thought to derive from the normal urothelial stem cells residing in the basal zone, recent studies suggest otherwise (16, 18). In particular, chemical carcinogenesis using a bladder-specific carcinogen, N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN), coupled with lineage tracing, suggests that low-grade noninvasive and high-grade MIUCB originate from intermediate and basal compartments, respectively (19, 20). It remains an open question, however, as to whether the different subtypes within MIUCB can also originate from different normal urothelial subtypes. Finally, are different MIUCB subtypes molecularly and phenotypically static or are they quite dynamic and interchangeable reflecting different stages of dedifferentiation and tumor progression? In other words, could the luminal subtype dedifferentiate and transition into the basal subtype during the course of tumor progression? Conversely, could the basal subtype regain the ability to differentiate into the luminal subtype thus becoming less aggressive subsequent to radio- and/or chemotherapy?

To begin to tackle some of these questions, we took an in-depth look of the effects of HRAS activation and p53 deficiency using a blend of in vitro and in vivo approaches. Activation of the RTK/RAS pathway and inactivation of the p53 pathway, events that were previously thought to define low-grade noninvasive and high-grade MIUCB, respectively (13, 21, 22), were recently found in whole-genome analyses to be equally prevalent in high-grade MIUCB (72% with RTK/RAS activation and 76% with p53 pathway activation; ref. 23). This suggests that alterations affecting both signaling pathways could overlap, simply by chance, in at least 50% of the MIUCB. One scenario is that this overlap is merely due to genetic drifting of two common events that do not necessarily cross-talk and are of no consequence to tumorigenesis. Another scenario is that these two events functionally converge as a result of selective pressure in tumor cells and that they collaborate or even synergize to exert a tumor-driving role leading to the formation of MIUCB. In this study, we examine these two competing hypotheses and our results have important implications on the molecular pathogenesis of MIUCB and shed light on how some of the MIUCB subtypes can be better managed clinically.

Materials and Methods

Transgenic, knockout, and compound mice

The transgenic mouse line, Upk2-HRAS*, harbored a single-copy transgene comprising a 3.6-kB murine uroplakin II promoter (UPII) and a constitutively active HRAS gene (24). The urothelial expression level of the HRAS* in this low-copy Upk2-HRAS* line is equivalent to that of the endogenous wild-type Ras, as evidenced by real-time PCR and Western blotting (24). The second transgenic line, Upk2-cre harbored a transgene comprising the UPII and a 1.4-kB cre recombinase gene (25). The third transgenic line harbored a “floxed” p53 allele (e.g., p53LOX) where loxP sites were inserted in introns 4 and 6, allowing deletion of exons 5 and 6 upon cre expression (26). The identity of Upk2-HRAS* and Upk2-cre was verified by Southern blotting and that of p53LOX by genomic PCR. Intercrosses were carried out among these three lines with additional crosses among their offspring, yielding a number of genotypes, from which four major genotypes were chosen for phenotypic characterization: (i) Upk2-cre (as negative control), (ii) Upk2-HRAS*/WT, (iii) Upk2-cre/p53LOX/LOX, and (iv) Upk2-HRAS*/WT/Upk2-cre/p53LOX/LOX. All animal experiments were approved by Institutional Animal Care and Use Committee.

Laser-capture microdissection and expression arrays

Since urinary bladders of Upk2-cre mice exhibited normal urothelia and those of Upk2-HRAS*/WT/Upk2-cre/p53LOX/LOX compound mice exhibited CIS and muscle-invasive lesions, these bladders were used for cross-sectioning and laser-capture microdissection. Briefly, 30 μm thick frozen sections were lightly stained with hematoxylin and the aforementioned lesions were dissected out using Leica LMD6000 Laser Micro-Dissection System. Total RNAs were extracted using RNeasy Micro Kit (Qiagen) and the RNA quality was verified by high-performance liquid chromatography. Microarray was carried out with Affymetrix 3′ IVT mouse expression arrays at our in-house facility (GEO accession number: GSE64756). Primary data were analyzed at the Center for Applied Genomics in University of Medicine and Dentistry of New Jersey and pathway and bioprocess analyses were performed online using Ingenuity iReport.

Cell culture, transfection, and establishment of stable lines

Human bladder urothelial carcinoma cell line, RT4, originally isolated from a low-grade, noninvasive urothelial carcinoma (27), was purchased from ATCC, maintained in McCoy's 5A medium containing 10% FBS and used within 6 months of receipt. Authentication of RT4 at ATCC used short tandem repeat profile and isoenzyme analysis. An shRNA of mouse p53 (5′-gactccagtggtaatctact-3′) was subcloned into retroviral vector, pMKO.1-puro (Addgene) and the resultant pMKO.1-puro/sh-p53 was cotransfected with pCL-10A1 packaging vector (Novus Biologicals) into cultured Phoenix cells. The packaged virus in the supernatant was collected and used to infect RT4 cells. Following a 10-day selection in culture medium containing 1 μg/mL puromycin, survived single clones were verified for p53 knockdown. HRASWT and HRASV12 were subcloned separately into retroviral vector, pBABE-hygro (Addgene), and cotransfected with the pCL-10A1 packaging vector into the Phoenix cells. The packaged retroviruses were isolated and infected into RT4 cells stably expressing the shRNA-p53. Stable clones were selected in culture medium containing 200 μg/mL hygromycin for 10 days and the resultant stable clones were verified for desired gene expression.

Cell migration and invasion assays

Cell migration of stable cell lines was first compared by wound-healing assay. When cultured cells reached 80% confluence, wounds were introduced under an inverted microscope using a sterile pipette tip. Wounded cells were cultured in fresh medium for 3 days before phase-contrast images were recorded. For invasion assay, BioCoat Matrigel Invasion Chamber (BD Biosciences) was used. Briefly, stable clones (2.5 × 104 cells) were seeded in 24-well chambers (in triplicate) containing 20 ng/mL 12-O-tetradecanoylphorbol-13-acetate. After incubation for 72 hours, the noninvading cells atop the membrane were removed by scrapping and, the invading cells underneath the membrane were visualized using Diff-Quik stain and counted in five high-power (×200) microscopic fields (one-center and four-peripheral).

Cell proliferation assay

Cell-cycle analysis

Urinary bladders were inverted to expose the mucosa. After incubation in a solution containing 1 mg/mL dispase at 4°C overnight, the urothelial cells were gently scraped off and digested with a solution containing 0.25% trypsin-EDTA at 37°C for 30 minutes. The cells were washed in PBS by centrifugation at 800 g for 5 minutes and filtered through a 100-μm pore size filter, fixed with precooled 70% ethanol at 4°C, and stained with 40 μg/mL propidium iodide containing 100 μg/mL RNase. Cell sorting was carried out using Facscan (Beckman) and the data were analyzed using ModiFit 3.2 (Verity Software House).

qRT-PCR

Total RNA was isolated from bladder urothelia using RNeasy Mini Kit (Qiagen) and 2 μg of it was used for cDNA synthesis using High Capacity cDNA Reverse Transcription Kit (Applied Biosystem). Real-time PCR was carried out with 7500 System (Applied Biosystems) under 95°C for 15′ for the first cycle; 95°C for 15″, 58°C for 20″ and 72°C for 30″ for 50 cycles, and 72°C for 5′ for the last cycle. PCR products were quantified by direct SYBR Green incorporation, with the relative abundance expressed as ratios to β-actin. The primers were: p19ARF-forward: gtcgcaggttcttggtcact, p19ARF-reverse: cgaatctgcaccgtagttga; p53-forward: agagaccgccgtacagaaga, p53-reverse: ctgtagcatgggcatccttt; p21-forward: cggtggaactttgacttcgt, p21-reverse: cagggcagaggaagtactgg.

Freshly dissected urinary bladders were fixed in PBS-buffered 10% formalin and embedded routinely in paraffin. Sections (4 μm) were stained with hematoxylin and eosin (H&E) for histologic examination. For IHC, deparaffinized sections were microwaved in a citrate buffer (pH 6.0) for 20 minutes to unmask the antigens and then incubated with primary (Supplementary Table S1) and secondary antibodies conjugated with horseradish peroxidase.

Statistical analysis

A Student t test (two tailed) was used to evaluate the statistical significance between Upk2-HRAS* mice and wild-type (Upk2-cre) mice in urothelial expression of p53 pathway components and between different groups of stably transfected cultured cells in their proliferation and invasion rates, with P value <0.05 considered statistically significant.

Results

A highly reproducible phenotype in transgenic mice bearing a single copy of oncogenic HRAS* under the control of the UPII promoter (Upk2-HRAS*) was the persistent urothelial hyperplasia (24). Compared with normal urothelium from the wild-type littermates (Fig. 1A, a), the hyperplastic lesions of the Upk2-HRAS* mice appeared as highly thickened, nonetheless well-differentiated urothelia with excellent polarity (Fig. 1A, b–d), and retention of superficial umbrella cells (arrows in Fig. 1A, c and d); and they started around 2 months of age and persisted through 12 months, without progressing, in grade or stage, to full-fledged urothelial tumor or reverting to normal urothelium. To understand the molecular underpinning of this phenomenon, we examined the cell-cycle status and found that at the steady state, there was a significant reduction of G0–G1 urothelial cells and increase of G2 cells in Upk2-HRAS* transgenic mice (12 months of age), as compared with the wild-type controls (Fig. 1B, top). This corresponded well with elevated mitogenic signals including phosphorylated ERK and AKT (both T308 and S473) in the transgenic mice (Fig. 1B bottom and Supplementary Fig. S1). However, S-phase cells were not significantly higher (Fig. 1B), suggesting that the DNA synthesis was held in check and that a prolonged G2 arrest existed, possibly due to concurrent induction of growth inhibitors/tumor suppressors (28). Of the tumor-suppressive pathways surveyed, that of p53, including p19, p53, and p21, exhibited marked upregulation on mRNA (Fig. 1C, left) and protein (Fig. 1C, right) levels. Such overt upregulation was not observed in pRB family proteins (e.g., pRB, p107, and p130). Interestingly, factors key to promoting G2–M transition such as CDC2 and CYCLIN B1 were significantly downregulated in the hyperplastic lesions of the Upk2-HRAS* mice (Fig. 1D), a phenomenon observed in nonurothelial cells with an upregulated p53 pathway (28). Our results suggest that oncogenic HRAS*-triggered proliferative forces are counter balanced by antiproliferative forces, especially by the p53 signaling axis, thus reaching an equilibrium and resulting in a nonprogressive and nonregressive state of persistent urothelial hyperplasia, that is quite different from oncogenic RAS-induced premature senescence and apoptosis in primary-cultured cells (29).

To determine whether the tumor-barrier effects of p53 upregulation by oncogenic HRAS* in urothelial cells were coincidental or causative, we introduced oncogenic HRAS* along with shRNA of p53 into cultured RT4 cells, which were originally derived from a human low-grade, superficial papillary bladder tumor (27) and lacked RAS or p53 mutation/deletion (30). Enforced expression of oncogenic HRAS* in RT4 elicited a marked upregulation of p53 and p21 (Fig. 2A). Knockdown of p53 or that along with the expression of a wild-type HRAS enhanced cell proliferation (Supplementary Fig. S2), but only slightly increased cell migration and invasion (Fig. 2C). In contrast, knocking down p53 and expressing an oncogenic HRAS* resulted in a dramatic increase of cell migration and invasion of RT4 cells (Fig. 2C and D). Thus, p53 deficiency and RAS activation appear to be synergistic in conferring the invasive property to human urothelial tumor cells and triggering the conversion of noninvasive human urothelial tumor cells into invasive ones.

To further define the interactive effects between oncogenic HRAS* and p53 deficiency in vivo, we developed compound mice by ablating p53 from urothelial cells expressing oncogenic HRAS*. To do so, we cross-bred three independent mouse lines: Upk2-HRAS* (24), Upk2-cre (in which the UPII drives the expression of a cre recombinase in urothelium; ref. 25), and floxed p53 (in which the exons of 5 and 6 were flanked by two loxP sites; Fig. 3A; ref. 26). We chose four resultant genotypes for phenotypic analyses: (i) Upk2-cre (as negative controls), (ii) Upk2-HRAS*/WT, (iii) Upk2-cre/p53LOX/LOX, and (iv) Upk2-HRAS*/WT/Upk2-cre/p53LOX/LOX (Fig. 3B and C). These four groups were followed for 16 months and, upon histopathological examination, the Upk2-cre and Upk2-cre/p53LOX/LOX lines exhibited normal urothelia, and the Upk2-HRAS*/WT line exhibited urothelial hyperplasia, as expected, throughout the 16-month observation (Fig. 3D). In stark contrast, the compound line expressing oncogenic HRAS* and lacking p53 developed exclusively high-grade bladder tumors in the form of CIS and muscle-invasive tumors (Fig. 3C and D and Fig. 4A–C and E–G). The invasive tumors arose as early as 6 months of age and, by 16 months, a majority (60%) of the mice harbored muscle-invasive bladder tumors (Fig. 3C). The CIS lesions were relatively flat with microinvasive lesions in adjacent lamina propria (Fig. 4B and C). The microinvasive and muscle-invasive lesions were confirmed by cytokeratin 5 staining (Fig. 4D, H, and I). These lesions bear strong resemblance to those found in human patients with muscle-invasive urothelial carcinoma, and lend strong support to the sequence of urothelial tumor progression from CIS to invasive tumors (13, 14, 31, 32). Finally, focal squamous differentiation within the muscle-invasive lesions was common as evidenced by H&E staining and by IHC staining using antibodies against keratin 1 and TRIM29 (Supplementary Fig. S3), markers for identifying squamous components in human muscle-invasive urothelial carcinomas (33).

That compound mice urothelially expressing oncogenic HRAS* and lacking p53 developed CIS and then invasive lesions also provided a unique opportunity for us to utilize these well-defined lesions to identify the molecular events that underlie this poorly defined progression step. Toward this end, we performed laser-capture microdissection of normal urothelia from the Upk2-cre mice, and CIS and muscle-invasive lesions from Upk2-HRAS*/WT/Upk2-cre/p53LOX/LOX compound mice. After high-quality mRNAs were isolated from freshly dissected lesions, the cDNAs were hybridized to oligonucleotide arrays representing all mouse genes (Affymatrix). Of differentially upregulated genes, those functioning in the EMT dominate the muscle-invasive lesions when CIS lesions were used as a reference (Table 1). Three groups of genes were particularly worth noting: (i) transcription factors that drive EMT [e.g., twist homolog 1 (TWIST), zinc finger E-box binding homeobox 2 (ZEB2), and ZEB1]; (ii) matrix-degrading enzymes (e.g., matrix metallopeptidases 13, 3, 2 and 9); and (iii) extracellular matrix components [e.g., collagen (type I, III, and IV), versican, vimentin, and fibronectin; Table 1]. Not surprisingly, those upregulated in the muscle-invasive tumor/CIS comparison were also upregulated in the muscle-invasive tumor/normal urothelium comparison. However, few of those upregulated in muscle-invasive tumors were also upregulated in the CIS lesions, indicating that the EMT genes are primarily switched on during muscle invasion. The only genes that showed more than 2-fold increase in CIS over normal urothelium were matrix metalloproteinase (MMP)-13 and platelet-derived growth factor receptor, suggesting their potential role(s) in CIS formation. Antibody staining confirmed that MMP2, 3, 9, and 13 were all overexpressed almost exclusively in the muscle-invasive lesions, with MMPs 2 and 9 primarily associated with tumor cells and MMP3 and 13 in both tumor cells and matrix (Fig. 5).

Upregulation of MMPs in muscle-invasive urothelial carcinoma cells. Immunochemical staining using anti-MMP antibodies followed by hematoxylin counterstaining was performed on bladder tissues from age-matched (8-months) Upk2-cre, Upk2-HRAS*, Upk2-cre/p53lox/lox, and Upk2-HRAS*/Upk2-cre/p53lox/lox mice. Note the significant upregulation of MMP2, MMP3, MMP9, and MMP13 in the muscle-invasive urothelial carcinoma cells of transgenic mice expressing the oncogenic HRAS* and deficient for p53. MMP3 and MMP13 were also detected strongly in some matrix cells. Magnification, ×200 for all panels.

Differential expression of genes important for EMT between MIUCB, CIS, and normal urothelium

EMT occurs in urothelial carcinoma progenitor/stem cells

To explore whether overexpression of EMT drivers occurred in more differentiated urothelial cells or in progenitor cells thus potentially playing a role in invasive tumor initiation, we performed IHC and double- and triple-immunofluorescent localization. Both ZEB1 and ZEB2 were strongly labeled in the nuclei of the invasive tumors cells of Upk2-HRAS*/WT/Upk2-cre/p53LOX/LOX compound mice, but were barely detectable in normal urothelia of Upk2-cre or Upk2-cre/p53LOX/LOX mice and were only weakly labeled in Upk2-HRAS* mice (Fig. 6A and B). Upon triple fluorescent staining, ZEB2 was found to be associated with cells specifically expressing CD44, a urothelial/carcinoma progenitor cell marker (Fig. 6C; refs. 18, 19, 34). Interestingly, these ZEB2- and CD44-positive cells had a marked decrease of E-cadherin, an epithelial marker (35), and a marked increase of vimentin, a mesenchymal cell marker (Fig. 6C; ref. 36). Double staining of ZEB2 with keratin 14, another urothelial progenitor cell marker (34), again showed excellent colocalization (Fig. 6D). Whereas normal-appearing urothelial regions showed K14-positive cells that lacked ZEB2 labeling, areas with tumor morphology showed strong coexpression of ZEB2 and K14 (Fig. 6D). Furthermore, areas with leading edge of invasion showed strong ZEB2 and K14 coexpression (Fig. 6D). Interestingly, invasive tumors cells of the Upk2-HRAS*/WT/Upk2-cre/p53LOX/LOX compound mice, but not the noninvasive cells of the single transgenic mice, strongly coexpressed in the nuclei activated AKT, activated β-catenin and ZEB2 (Supplementary Fig. S4), suggesting this signaling pathway as the underlying cause of EMT activation. These results establish that urothelial tumor progenitor cells in our compound transgenic mice expressing oncogenic HRAS* and lacking p53 strongly express EMT drivers and their expression may play a central role in initiating muscle-invasive urothelial carcinoma. Finally, in contrast with the expansion of K14-positive cells in the muscle-invasive lesions, cells positive for keratin 20, a marker expressed in urothelial superficial umbrella cells and used for terminal differentiation of normal urothelium (37), were completely absent from the muscle-invasive lesions (Supplementary Fig. S5). These results, together with our observation of focal squamous differentiation of the muscle-invasive lesions, strongly indicate that the muscle-invasive urothelial carcinoma of the bladder that we observed in our Upk2-HRAS*/Upk2-cre/p53LOX/LOX mice belongs to the “basal-subtype” recently classified in patients (4–6, 8–10).

Detection of transcriptional factors driving EMT in urothelial progenitor cells. A and B, urinary bladders from age-matched (8-months) Upk2-cre, Upk2-HRAS*/WT, Upk2-cre/p53lox/lox, and Upk2-HRAS*/WT/Upk2-cre/p53lox/lox mice were immunohistochemically stained with anti-ZEB1 (A) and anti-ZEB2 (B) and counterstained by hemotoxylin. Note the marked upregulation of both proteins almost exclusively in the muscle-invasive lesions of the Upk2-HRAS*/WT/Upk2-cre/p53lox/lox mice. C, urinary bladders from Upk2-HRAS*/WT and Upk2-HRAS*/WT/Upk2-cre/p53lox/lox mice were triple stained using immuofluorescent method with anti-E-cadherin (E-cad), -ZEB2, and -CD44 (left two) or with anti-vimentin, -ZEB2, and -CD44 (right two). DAPI was used to visualize the nuclei. Note the marked downregulation of E-cadherin and dramatic upregulation of ZEB2 in CD44-positive cells in the muscle-invasive lesions of the Upk2-HRAS*/WT/Upk2-cre/p53lox/lox mice. Also, note the colocalization of vimentin, ZEB2, and CD44 in the invasive tumor cells (far-right, arrows). D, urinary bladders from Upk2-HRAS*/WT, Upk2-cre/p53lox/lox, and Upk2-HRAS*/WT/Upk2-cre/p53lox/lox mice were subject to immunofluorescent staining with anti-ZEB2 and -keratin 14 antibodies, with DAPI as counterstaining to visualize the nuclei. Note the lack of ZEB2 staining in K14-positive cells in Upk2-HRAS*/WT and Upk2-cre/p53lox/lox mice and the strong staining of ZEB2 in K14-positive cells in Upk2-HRAS*/WT/Upk2-cre/p53lox/lox mice (middle, dashed circle). Dashed box illustrates an area of normal-appearing urothelium, showing the lack of ZEB2 staining. Also note that the leading edge of an early invasive lesion in Upk2-HRAS*/WT/Upk2-cre/p53lox/lox mice had marked upregulation of ZEB2 in K14-positive cells (right). Magnification, ×200 for all panels.

Discussion

The recent expansion of whole-genome and whole-exome sequencing into a broad range of human cancers has yielded unprecedented details about somatic gene mutations, making it possible to classify cancers in genomic terms and to devise target-specific, precision therapies (38). Urothelial carcinoma of the bladder (UCB) is no exception. In a landmark paper (23), The Cancer Genome Atlas (TCGA) Research Network reported a comprehensive, multiplatform analysis of 131 high-grade, MIUCB on their somatic mutation, DNA copy number, messenger and miRNA expression, protein and phosphorylated protein expression, and DNA methylation. Of the several surprises from that report, one relates to the high frequency of alterations in the RTK/RAS/PIK3K signaling axis. Up to 72% of the high-grade MIUCB harbored activation mutations in the FGFR3, EGFR, ERBB2, ERBB3, HRAS/NRAS, and PIK3CA or inactivating mutations in NF1, PTEN, INPP4B, STK11, TSC1, and TSC2 (23). This is surprising because alterations in this pathway were previously assigned primarily to low-grade, noninvasive UCB and to predict low risk of progression and favorable clinical outcome (13, 21, 22), a concept supported by independent studies using genetically engineered mice. For instance, urothelial expression of an FGFR3 mutant (K644E) that constitutively activates the tyrosine kinase of FGFR3, either alone or in combination with KRAS and β-catenin mutations or with PTEN deletion, in transgenic mice failed to elicit any urothelial carcinoma (39). Similarly, urothelial overexpression of an EGFR in our transgenic mice induced proliferation but not tumor formation even after an exhaustively long (28-month) follow-up (40). Furthermore, urothelium-specific expression in our transgenic mice of oncogenic HRAS* at a level comparable with the endogenous RAS elicited urothelial hyperplasia that only occasionally progressed to low-grade, papillary noninvasive UCB in aged mice (>12 months; ref. 24). High-grade MIUCB was never observed in any of these RTK/RAS pathway-activated mouse models (24, 39, 40). The fact that gene mutations that activate the RTK/RAS pathway are highly prevalent in human high-grade MIUCB from the TCGA study (23) raises an important question as to whether these mutations are tumor “drivers” or “passengers” and whether the mutations require additional genetic alterations to be tumorigenic.

Our present study provides experimental evidence establishing that RAS activation per se is nontumorigenic in urothelial cells in vivo due, in large part, to a compensatory tumor barrier that RAS elicits in the p53 tumor suppressor pathway (Figs. 1 and 2 and Supplementary Fig. S1). Although p53 deficiency by itself is also nontumorigenic, it is highly synergistic with RAS activation, and these two alterations together are necessary and sufficient to initiate high-grade, CIS and MIUCB (Figs. 3 and 4). Of note, the MIUCB we observed in our double transgenic mice expressing oncogenic Ha-RAS and lacking p53 bears strong resemblance to the basal subtype of MIUCB recently classified in patients (4–11) in their (i) high expression of basal cell markers such as K5, K14, and CD44 (Figs. 4 and 6 and Supplementary Fig. S5); (ii) low or lack of expression of luminal cell markers such as E-cadherin and K20 (Fig. 6 and Supplementary Fig. S5); (iii) focal squamous differentiation (Supplementary Fig. S3); and (iv) high expression of EMT transcription factors (Twist, ZEB1, and ZEB2; Table 1; Fig. 6), EMT markers (vimentin, MMPs 2, 3, 9, and 13; Table 1; Figs. 5 and 6), and extracellular matrix components (collagen, versican, and fibronectin; Table 1). Our study therefore functionally defines RAS pathway activation and p53 deficiency as the highly synergistic codrivers for the basal-subtype MIUCB, and it has several significant implications. First, as has been demonstrated in other cancer types, tumor drivers (as opposed to the passengers) are more reliable biomarkers for cancer subclassification and prediction of chemotherapeutic response and clinical outcome (12). RAS pathway activation together with p53 deficiency could potentially serve as a new biomarker set for the genetic identification of the basal subtype of MIUCB that may be associated with an unfavorable prognosis, hence requiring aggressive therapeutic modalities. Second, our study reveals a previously unrecognized molecular cross-talk between RAS and p53 pathways in converting low-grade noninvasive urothelial lesions (e.g., hyperplasia and low-grade papillary) into becoming high-grade noninvasive (e.g., high-grade papillary and CIS) and invasive ones (e.g., MIUCB). Not only did we demonstrate such a relationship in transgenic mice (Figs. 3–6; Supplementary Figs. S3–S5), but we also showed that introducing oncongenic HRAS* and knocking down p53 in cultured RT4 cells confer invasive properties to these otherwise noninvasive human UCB cells (Fig. 2). It has been suggested, based on clinical longitudinal studies, that approximately 25% of the low-grade, noninvasive UCB can eventually progress in grade and/or stage to muscle invasion (41, 42). This occurs in an unpredictable manner that necessitates lifelong, vigilant follow-up by repeated cystoscopy and biopsy, a main cause for morbidity, time lost from work and high medical expenses. Thus far, no biomarker exists that can reliably predict the risk of progression of noninvasive UCB to the invasive stage (43, 44). Perhaps it is not surprising that p53 alterations are not very predictive of UCB progression (45), based on data from genetically engineered mice indicating the lack of tumorigenicity by p53 deficiency alone (Fig. 3; refs. 26, 46, 47). It is possible, however, that a combination of RAS pathway activation and p53 deficiency, as we demonstrated here, are better biomarkers for UCB surveillance and prediction of tumor progression. It is worth noting that ablation of both PTEN and p53 in mouse urothelia also led to MIUCB (48), consistent with the fact that PTEN acts in the RAS pathway and PTEN inactivation is functionally akin to RAS activation. Third, because RAS activation is a codriver of the basal-type MIUCB, inhibition of this pathway might be of significant value in treating and/or preventing the progression of this MIUCB subtype. The fact that the basal-type MIUCB in humans is often resistant to the existing chemotherapeutics (4) makes RAS pathway inhibition a particularly attractive avenue to explore. Because suppressing activated RAS per se remains challenging (49), it is likely that effectors of RAS will have to be targeted and that inhibition of more than one signaling branch (e.g., PI3K-AKT as well as MAPK) is required to achieve satisfactory results (50). Finally, the development of a new transgenic mouse model that consistently develops the basal-type MIUCB provides a novel in vivo platform for dissecting the evolutionary steps and the potential cross-talks among the different MIUCB subtypes and for testing subtype-specific diagnostic, preventive and therapeutic strategies. Clearly, many of these ideas require clinical validation studies before they can be translated to the bedside.

From a mechanistic standpoint, RAS activation and p53 deficiency could synergize on several fronts to affect cellular processes that govern urothelial tumorigenesis and progression. As shown recently, RAS activation increases the replicative pressure on urothelial cells, causing them to undergo DNA damage (51). Under normal circumstances, that is, when p53 pathway is intact, urothelial cells can sense DNA damage and upregulate p19Arf, which in turn upregulates p53 and downstream effectors such as p21 (Fig. 1). This helps restrain G1–S and G2–M transition and allow time for DNA damage repair to take place. When p53 pathway is defective, however, cell-cycle progression proceeds with amplification of the damaged DNA, setting a stage for malignant transformation. Another level of interaction is the collaborative nature of RAS activation and p53 deficiency on cell motility. Activated RAS is a strong enhancer of cell motility (52), whereas a functional p53 is a potent cell motility inhibitor (53). As we showed in our in vitro assay, activated RAS or p53 knockdown alone only had a marginal increase on cell motility, but combining these two events resulted in a marked increase of cell motility and triggered invasion (Fig. 2). Finally, as with other epithelial cells, RAS activation and p53 deficiency are both strong promoters of EMT (54, 55). The MAPK and AKT pathways, both shown to be prominently activated in our transgenic mice (Fig. 1 and Supplementary Fig. S1), can activate factors such as β-catenin that drive EMT (Table 1 and Supplementary Fig. S4). While normal p53 negatively regulates this process, p53 deficiency fuels EMT and sets the tumor cell invasion in motion (Figs. 2–6 and Supplementary Figs. S3–S5). There is mounting evidence suggesting that EMT can lead to drug resistance (35). Since EMT enhances the stemness and the plasticity of urothelial cells, it may also fuel the trans-differentiation of some of the urothelial progenitor cells toward the squamous lineage and squamous differentiation, another potential cause of drug resistance. In this regard, inhibiting RAS effectors that drive EMT and/or inhibiting EMT effectors such as MMPs may play a critical role in reducing chemoresistance that has been observed in the basal-type MIUCB (4). Because EMT is highly activated in progenitor/stem cells that give rise to the basal-type MIUCB (Fig. 6 and Supplementary Fig. S5), its suppression may present a unique opportunity for controlling the root cause of tumor cell expansion and invasion.

In summary, the data presented in this paper provide the first experimental evidence demonstrating that the loss of p53 is critical in allowing hyperplastic urothelial cells in vivo to bypass G2 arrest induced by activated HRAS and proceed to tumor formation; that RAS pathway activation and p53 pathway inactivation together confer invasive properties to noninvasive urothelial tumor cells and these two synergistic events are necessary and sufficient to convert CIS to basal-subtype, MIUCB; and that activation of EMT and increased stemness in urothelial progenitor cells are crucial epigenetic events for invasive tumorigenesis. Our data also strongly suggest that increased urothelial plasticity due to EMT may underlie urothelial trans-differentiation to the squamous lineage, leading to focal squamous differentiation in urothelial carcinomas. From a clinical standpoint, combined RAS pathway activation and p53 pathway inactivation, events highly prevalent in human urothelial carcinomas as evidenced by whole-genome analyses, may serve as a new biomarker set to predict urothelial carcinoma progression, and inhibition of receptor tyrosine kinase/RAS pathway components may be used as therapeutic targets for basal-subtype, muscle-invasive urothelial carcinomas that are resistant to conventional chemotherapeutics.

Grant Support

This work was supported in part by grants from the United States NIH (P01 CA165980) and Veterans Affairs Office of Research and Development (Biomedical Laboratory Research and Development Service; 1I01BX002049) and a grant-in-aid from the Goldstein Fund for Urological Research of the New York University School of Medicine.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Footnotes

Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

References

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